New chemical agents based on adamantane-monoterpene conjugates against orthopoxvirus infections

Article abstract of DOI:10.1039/d0md00108b

Currently, the spectrum of agents against orthopoxviruses, in particular smallpox, is very narrow. Despite the fact that smallpox is well controlled, there is, for many reasons, a real threat of epidemics associated with this or a similar virus. In order to search for new low molecular weight orthopoxvirus inhibitors, a series of amides combining adamantane and monoterpene moieties were synthesized using 1- and 2-adamantanecarboxylic acids as well as myrtenic, citronellic and camphorsulfonic acids as acid components. The produced compounds exhibited high activity against the vaccinia virus (an enveloped virus belonging to the poxvirus family), which was combined with low cytotoxicity. Some compounds had a selectivity index higher than that of the reference drug cidofovir; the highest SI = 1123 was exhibited by 1-adamantanecarboxylic acid amide containing the (-)-10-amino-2-pinene moiety. The produced compounds demonstrated inhibitory activity against other orthopoxviruses: cowpox virus (SI = 30-406) and ectromelia virus (mousepox virus, SI = 39-707). This journal is

Full text of DOI:10.1039/d0md00108b

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RESEARCH ARTICLE
New chemical agents based on adamantane–
monoterpene conjugates against orthopoxvirus
infections†
Cite this: DOI: 10.1039/
d0md00108b
a Evgenii S. Mozhaytsev,a Dina V. Korchagina,a
*
Evgenii V. Suslov,
ac
Nikolay I. Bormotov,b Olga I. Yarovaya,
Konstantin P. Volcho,ac Olga A. Serova,b
Alexander P. Agafonov,b Rinat A. Maksyutov,b
Larisa N. Shishkinab and Nariman F. Salakhutdinovac
Currently, the spectrum of agents against orthopoxviruses, in particular smallpox, is very narrow. Despite
the fact that smallpox is well controlled, there is, for many reasons, a real threat of epidemics associated
with this or a similar virus. In order to search for new low molecular weight orthopoxvirus inhibitors, a
series of amides combining adamantane and monoterpene moieties were synthesized using 1- and
2-adamantanecarboxylic acids as well as myrtenic, citronellic and camphorsulfonic acids as acid
components. The produced compounds exhibited high activity against the vaccinia virus (an enveloped
virus belonging to the poxvirus family), which was combined with low cytotoxicity. Some compounds had
a selectivity index higher than that of the reference drug cidofovir; the highest SI = 1123 was exhibited by
1-adamantanecarboxylic acid amide containing the (−)-10-amino-2-pinene moiety. The produced
compounds demonstrated inhibitory activity against other orthopoxviruses: cowpox virus (SI = 30–406)
and ectromelia virus (mousepox virus, SI = 39–707).
Received 6th April 2020,
Accepted 30th June 2020
DOI: 10.1039/d0md00108b
rsc.li/medchem
smallpox remains. Second, the development and accessibility
of biotechnologies pose a threat of reproduction of the VARV
Introduction
In 1980, the World Health Organization (WHO) Assembly
declared eradication of black (or natural) smallpox, caused by
the agent referred to as the variola virus (VARV), which
belongs to the genus Orthopoxvirus, on the planet. Since that
time, vaccination against smallpox has been discontinued.
Smallpox was the first disease in human history to be
controlled by means of mass vaccination. Now, about 50% of
the world's population is estimated to have no smallpox
immunity. However, an Independent Advisory Group on
Public Health Implications of Synthetic Biology Technology
Related to Smallpox report to the WHO Director-General
noted the need to continue developing new low molecular
weight agents against the VARV.1 This conclusion is related to
several reasons. First, due to climate change, the risk of
smallpox spread from permafrost soils containing human
or a similar virus for terrorist purposes.2 Also, there is a risk
of illegal SV storage and deliberate use of natural or
recombinant VARV strains against the population. Another
argument in favor of the development of new anti-
orthopoxvirus agents is the potential danger to humans of
other orthopoxviruses circulating in animal populations, such
as monkeypox viruses and cowpox viruses, which evolve,
spread, and periodically cause disease outbreaks in humans.
a Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian
Academy of Sciences, Lavrentjev Avenue 9, 630090 Novosibirsk, Russia.
E-mail: suslov@nioch.nsc.ru
b Department of Prevention and Treatment of Especially Dangerous Infections, State
Research Center of Virology and Biotechnology VECTOR, Koltsovo, Novosibirsk
Region, 630559, Russian Federation
c Novosibirsk State University, Pirogova St. 2, 630090 Novosibirsk, Russia
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00108b
Fig.
replication.
1 Chemical structure of the inhibitors of orthopoxvirus
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For example, the latest monkeypox outbreak in humans
occurred in Africa in 2016;3 it is worth noting that mortality
from this disease amounts to 17%.4,5
The only drug available for the treatment of smallpox and
monkeypox is tecovirimat (TPOXX®), created on the basis of
chemical compound ST-246 developed by SIGA Technologies
Inc. (USA) (Fig. 1), was approved by the Food and Drug
Administration (FDA) for use in the United States in 2018.6
The mechanism action of tecovirimat (TPOXX®, ST-246) is
different from that of cidofovir (CDV) that inhibits viral DNA
replication. The target of chemical compound ST-246 is a
highly conserved virus-encoded protein, p37, present in all
orthopoxviruses.7 An agent NIOCH-14, an analogue of ST-246,
was developed at the Novosibirsk Institute of Organic
Chemistry in cooperation with the VECTOR State Research
Center of Virology and Biotechnology. This compound has
been effective against orthopoxviruses in in vivo and in vitro
experiments.8 The mechanism of action of NIOCH-14 is the
same as that of ST-246 because NIOCH-14 is its prodrug and
metabolized into ST-246 in mammals. Another anti-smallpox
drug, brincidofovir (CMX001), which is undergoing clinical
Fig. 2 Chemical structure of inhibitors of orthopoxvirus replication
containing adamantane 1–3 and 1,7,7-trimethylbicyclo[2.2.1]heptane
4–8 scaffolds.
compounds towards other orthopoxviruses has not been
studied.
trials,9 is
a lipophilic nucleotide analogue of cidofovir
(Fig. 1). Cidofovir is an antiviral drug used for treating
cytomegalovirus retinitis; in this case, cidofovir exhibits
activity in lethal orthopoxvirus infection models in mice and
monkeys.10 Cidofovir was shown to have low oral
bioavailability and shown to be toxic to the kidneys; in
addition, this drug was not effective when used after
manifestation of smallpox lesions in VARV-infected monkeys.
In the case of brincidofovir in small rodent models, it does
not provide an adequate level of protection against lethal
ectromelia infection in mice.11 Nevertheless, potential
development of orthopoxvirus resistance to brincidofovir and
tecovirimat necessitates further research for creating new
biologically active compounds with mechanisms of action
differing from those of drugs that are at the advanced stages
of development, as it was concluded by the Advisory Group of
Independent Experts to Review the Smallpox Programme in
comments12 to the WHO report “Scientific review of variola
virus research, 1999–2010”.10
Natural compounds, in particular natural bicyclic
monoterpenoids containing the bicyclo[2.2.1]heptane moiety,
have been used as starting molecules for the development of
antiviral drugs.17 For example, earlier we discovered highly
effective influenza virus inhibitors based on camphor,18
borneol,19 myrtenol20 and isopulegol,21 and showed high
anti-filovirus activity of compounds containing the 1,7,7-
trimethylbicyclo[2.2.1]heptane scaffold.22,23 Expanding the
spectrum of antiviral activity of the synthesized agents, we
have recently started studying their properties against
vaccinia viruses. Thus, we demonstrated that ester 4 and
amide 5 exhibited pronounced activity against the vaccinia
virus.24 Camphor-derived compound 6 (Fig. 2) containing a
thiazole ring showed activity in a lower micromolar range
(IC50: 2.4–3.7 μM; IC50 is the virus inhibitory concentration
providing 50% cell survival in virus-infected monolayers) with
moderate cytotoxicity in the Vero cell line (CC50: 64–94 μM;
CC50 is the toxicity concentration causing 50% cell death in
uninfected monolayers).25 Camphor N-acylhydrazones 7 and 8
showed an even higher selectivity index (Fig. 2); their IC50
and CC50 were 5.1 μM and 1430 μM for 7 and 6.2 μM and
490 μM for 8, respectively.26 Agents 4–8 described by us were
an order of magnitude more active than the reference drug
cidofovir (IC50: 40 μM). Previously, we found that connection
Some adamantane derivatives are known to exhibit
antiviral activity, mainly against the influenza virus.13
However, a vaccinia virus inhibitor, 1H-indole-2,3-dion-3-(N-2-
adamantyl thiosemicarbazone) 1, was found among
adamantane derivatives (Fig. 2); exposure of the cell culture
to the inhibitor at a concentration of 2 μg ml−1 for 3 h
reduced vaccinia virus reproduction by 42%.14 Some
of
a monoterpene moiety with an aminoadamantane
substituted
aminoacetyl-adamantylamines
also
have
molecule increased its antiviral properties against
a
pronounced inhibitory activity against the vaccinia virus (e.g.,
compound 2, Fig. 2).15 A study16 demonstrated that N-1-
adamantyl-2,2-diphenylacetamide 3 (Fig. 2) at a concentration
of 2 μg ml−1 reduced reproduction of the vaccinia virus in cell
culture by 72 to 100%. However, these studies lack the data
on toxicity of the compounds, which prevents assessment of
the selectivity index and, accordingly, the practical potential
of these compounds. In addition, activity of the presented
rimantadine-resistant strain of the A(H1N1)pdm09 influenza
virus.27 At the same time, the anti-smallpox activity of
compounds combining adamantane and monoterpene
moieties was not previously studied.
The purpose of this study was to synthesize amides
combining adamantane and monoterpene moieties to
investigate
their
inhibitory
activity
against
orthopoxviruses.
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Results and discussion
Chemistry
At the first stage, several monoterpene amines were
synthesized for producing target amides containing an
adamantane moiety. For this purpose, bromo derivatives
16–21 were prepared from (−)-myrtenol 9, (−)-nopol 10, (−)-
perillyl alcohol 11, 3,7-dimethyloctanol 12, geraniol 13, and
nerol 14 by the reaction with PBr3 (Scheme 1). Almost all
bromides were isolated from the reaction mixtures by column
chromatography in yields of 30 to 95%. Bromides 20 and 21
were unstable on SiO2 and were used without purification.
Bromide 22 was synthesized from (S)-citronellol 15 by
treatment with N-bromosuccinimide in the presence of PPh3
according to the procedure in ref. 28 (Scheme 1). The yield of
compound 22 after purification by column chromatography
on silica gel amounted to 79%. Amines 23–29 were produced
from bromides 16–22 according to the Gabriel procedure
(Scheme 1). Amines 23, 24 and 26–28 were previously
described in ref. 29–35.
By using (+)-camphor 30, (−)-fenchone 31, and (+)-
campholenic aldehyde 32, we prepared appropriate oximes
33–35 (Scheme 2), which were then reduced to amines using
the NaBH4–NiCl2 system for compounds 36 and 37, and
LiAlH4 was used as a reducing agent for oxime 35. In this
case, amines 36 and 37 were formed as mixtures of exo- and
endo-isomers; predominant diastereomers were isolated from
the reaction mixtures by column chromatography.
Scheme 2 Reagents and conditions: (i) NH2OH·HCl, NaOAc, MeOH/
H2O, reflux; (ii) NaBH4, NiCl2, −40 °C; (iii) NH2OH·HCl, K2CO3, EtOH, 3
h; (iv) LiAlH4, THF, reflux, 3 h.
1-Adamantanecarboxylic
acid
chloride
39
is
a
commercially available reagent. Its isomer, 2-adamantane
carboxylic acid chloride 52, was synthesized using
2-adamantanone 50. The method of synthesis of acid
chloride 52 included preparation of an appropriate oxirane
by reacting 50 with trimethylsulfoxonium iodide,37 opening
of the epoxide group to form the aldehyde, and oxidizing
the resulting aldehyde to acid 51 using the Jones reagent.38
2-Adamantane carboxylic acid chloride 52 was prepared
according to the procedure39 by boiling acid 51 in SOCl2
(Scheme 4). Another series of amides 53–56 were prepared
by reacting acid chloride 52 with amines 23, 24, 26, and 38
(Scheme 4).
Given the commercial availability of citronellic acid 57,
the appropriate acid chloride was synthesized, the interaction
of which with 1- and 2-aminoadamantanes gave amides 58a
and 58b (Scheme 5). Chromatographic purification of the
compounds resulted in mixtures containing, along with
amides 58a or 58b, starting acid 57. Treatment of the
resulting mixtures with a NaOH solution was found to not
remove the acid. However, replacement of sodium hydroxide
with potassium hydroxide led to complete removal of the
The reaction of amines 23–29 and 36–38 with
1-adamantanecarboxylic acid chloride 39 was used to prepare
target amides 40–49 in yields from 12% to 99% (Scheme 3).
In some cases, preparation of compounds with purity
suitable for biological studies required additional
purification of the resulting amides by column
chromatography or recrystallization, which decreased their
yields, e.g., in the case of amides 40–42 and 44–46.
Compounds 43, 45, and 46 were previously described by
Chepanova et al.36
Scheme 1 Reagents and conditions: (i) was used for 16–21: PBr3 (0.4
eq.), Et2O; (ii) was used for 22: NBS, PPh3, CH2Cl2; (iii) potassium
phthalimide (1.1 eq.), DMF, 60 °C, 6 h; (iv) ethylenediamine (2 eq.),
MeOH, reflux, 4 h.
Scheme 3 Synthesis of compounds 40–49.
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Biology
Compounds were screened for virus inhibitory activity (IC50
)
against orthopoxviruses using the vaccinia virus (Copenhagen
strain) in Vero cell culture; cytotoxicity (CC50) of the
compounds was studied in the same cell culture (Table 1).
Cytotoxicity and inhibitory activity were assessed in Vero cell
culture using an adapted colorimetric method.41 As positive
controls, i.e. agents with established antiviral activity against
Scheme
4
Reagents and conditions: (i) Me3SO+I−, NaOH, iPrOH,
reflux; (ii) BF3·Et2O, benzene, stirring, 1 min; (iii) CrO3/H2SO4/H2O,
Et2O; (iv) SOCl2, reflux, 4 h; (v) corresponding amine (1 eq.), Et3N (1.1
eq.), dry toluene, 0 °C.
orthopoxviruses,
two
compounds
were
used:
the
commercially available drug cidofovir (Heritage Consumer
Products, USA) and ST-246, synthesized for research purposes
in the Novosibirsk Institute of Organic Chemistry according
to the method described by the authors.42 These anti-
orthopoxvirus drugs, which exhibit different mechanisms of
action, were used not only for comparison, but as verified
internal standards to confirm the normal functioning of the
virus–cell system for testing the activity of the newly studied
drugs.
An analysis of the data presented in Table 1 shows that
amides containing acyclic monoterpene substituents
(compounds 43–46, 55, 58a, and 58b) mainly exhibited
moderate inhibitory activity or no activity against the vaccinia
virus (IC50: 2.8–14.8 μM) and fairly high toxicity to Vero cells
(CC50: 12.9–37.0 μM), which led to low selectivity indices (SI).
Amide 58b derived from citronellolic acid and
2-aminoadamantane was the least cytotoxic compound in this
group (CC50: 330 μM), which, in combination with an IC50
value of 14.8 μM, led to a SI of 22. The results comparable to
those of compound 58b were also obtained for a monocyclic
perillyl alcohol monoterpene derivative 42. Amides 43 and 45
containing 3,7-dimethyloctanol and nerol moieties had no
activity against the vaccinia virus, but were highly toxic (CC50:
17 μM and 13 μM, respectively).
Scheme
5 Reagents and conditions: (i) NaClO2, KH2PO4, H2O2,
Transition
to
compounds
containing
a
(−)-
t-BuOH; (ii) SOCl2, dry benzene as a solvent, reflux, 4 h; * without
solvent (iii) corresponding amine (1 eq.), Et3N (1.1 eq.), dry toluene, 0
campholenaldehyde moiety, amides 49 and 56, demonstrates
that compounds 49 and 56 having activity comparable to that
of derivatives containing acyclic monoterpenoid substituents
exhibited lower cytotoxicity, which led to a good SI of about
126 and 122, respectively, with the latter being independent
of the amide group location in the adamantane scaffold.
Among the studied compounds, the most promising
group was that containing bicyclic monoterpenoid moieties
((−)-10-amino-2-pinene: 40, 41, 53, 54, 61a, and 61b; camphor:
47, 63a, and 63b; and fenchone: 48). In this group, there were
amides with a maximum selectivity index, such as compound
40 with a SI of 1123.
Comparison of amides 40, 53, 41, and 54 reveals that an
increase in the length of the linker between adamantane and
monoterpene moieties increases the cytotoxicity of the
compounds. Furthermore, as follows from Table 1,
1-adamantanecarboxylic acid derivatives have higher activity
than 2-adamantanecarboxylic acid derivatives. Introduction of
camphor or fenchone as substituents decreases the activity of
the compounds (i.e. increases IC50), as in amides 47 and 48.
Transition to sulfamides 63a and 63b reveals that the activity
°C.
residual acid, as a salt, into the aqueous phase. Compounds
58a and 58b were obtained in yields of 70% and 72%,
respectively.
Myrtenic acid chloride was prepared by oxidation of (−)-
myrtenal 59 to the appropriate acid 60 with sodium chlorite
under slightly acidic conditions, according to the procedure
in ref. 40 (Scheme 5) and subsequent boiling with SOCl2. Its
interaction with 1- and 2-aminoadamantanes provided
appropriate amides 61a and 61b (Scheme 5) in yields of 71%
and 30%, respectively.
Sulfonamides 63a and 63b were synthesized from
camphorsulfonic acid 62 according to a procedure similar to
that used for the preparation of citronellic acid amides,
which involved the synthesis of an appropriate acid chloride
and its interaction with aminoadamantanes (Scheme 5). The
yields of compounds 63a and 63b were 50% and 34%,
respectively.
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Table 1 Indicators of cytotoxicity (CC50) and antiviral activity (IC50) of
amides 40–49, 53–56, 58a, 58b, 61a, 61b, 63a and 63b against the
vaccinia virus (VV) in Vero cell culture
Table 1 (continued)
Compound
CC50, μM
IC50, μM
SI
316.7 39.3
2.6 0.2
122
Compound
CC50, μM
IC50, μM
SI
1908.6 101.4
1.7 0.2
1123
56
37.0 4.6
3.1 0.2
12
40
41
750.2 84.6
319.0 39.6
1.8 0.2
417
26
58a
58b
329.5 40.8
7.9 0.9
14.8 0.9
0.7 0.1
22
11
12.3 0.8
42
43
44
17.3
na
—
3
61a
61b
811.5 110.5
164.2 20.3
2.5 0.1
2.3 0.1
325
71
18.9 2.3
12.9
5.9 0.6
na
—
63a
95.8 11.8
0.6 0.04
160
45
46
18.1 2.2
3.6 0.2
5
63b
Cidofovir
ST-246
475.3 30.1
1276 202
40.0 1.2
0.01 0.003
12
127 600
Note: CC50 is the cytotoxic concentration causing 50% cell death in
an uninfected monolayer; IC50 is the inhibitory concentration
ensuring 50% cell survival in a virus-infected monolayer; na – no
activity, no IC50 values were found for these compounds; as M SD,
where M is the mean, and SD is the standard deviation; n = 3 is the
number of CC50 and IC50 measurements; SI is the drug selectivity
index (CC50/IC50).
316.7 39.3
10.6 0.7
30
47
48
316.9 39.3
316.9 39.3
14.4 0.9
2.5 0.2
22
126
of these compounds increases compared to that of amides 47
and 48, but compounds 63a and 63b are more cytotoxic than
47 and 48. Unlike amides 40, 45, 41, and 54, higher activity
among compounds 63a and 63b was demonstrated by the
substance with a substituent at position 2 of the adamantane
moiety. 2-Aminoadamantane derivative 61b has a selectivity
index an order of magnitude higher than that of compound
61a, which is mainly due to the high cytotoxicity of amide
61a. In this case, almost all amides containing bicyclic
monoterpenoid substituents showed higher activity (IC50:
0.6–14.4 μM) than the reference drug cidofovir (IC50: 40 μM),
and their selectivity index exceeded the SI of the reference
drug by two orders of magnitude. For amides that are
structurally similar and could be considered as amides
containing an amide bond with a reversed direction, different
trends were found. In general, transition to amides of
monoterpene acids 58a, 61a, 61b leads to slightly increased
49
53
1225.9 143.9
372.5 97.7
4.6 0.2
4.4 0.1
267
85
54
55
13.7 1.7
2.8 0.2
5
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activity compared to derivatives of adamantane carboxylic
acids 40, 46, 53. At the same time, the derivatives of myrtenic
acid 61a and 61b showed higher cytotoxicity than their
analogs – amides, containing myrtenic fragment 40 and 53.
On the other hand, the amide of citronellic acid 58a was
found to be about 2 times less cytotoxic than 1-adamantane
carboxylic acid derivative 46.
Therefore, in this study, we discovered a new type of
highly effective low-toxic orthopoxvirus inhibitors combining
monoterpenoid and adamantane moieties via an amide
linker.
Conclusions
In
summary,
bicyclic
monoterpene
derivatives
In this study, we investigated a series of new amides for their
inhibitory activity against orthopoxviruses (vaccinia virus –
VV, cowpox virus – CPXV, mousepox virus – ectromelia virus –
ECTV). For this purpose, we synthesized a series of new
amides 40–49 and 53–56 combining adamantane and
monoterpene moieties; they were prepared starting from
monoterpene amines 23–29 and 36–38, which were then
acylated with 1- and 2-adamantanecarboxylic acid chlorides.
Some amides (58, 59, 61a, 61b, 63a, and 63b) were also
prepared starting from 1- and 2-aminoadamantanes. Almost
all amides containing bicyclic (β-bornane and pinene)
monoterpenoid substituents exhibit higher activity (IC50:
0.59–14.40 μM) than the reference drug cidofovir (IC50: 40
μM), and their selectivity index exceeds that of the reference
drug by two orders of magnitude towards the vaccinia virus.
The highest activity in combination with low cytotoxicity was
exhibited by compounds containing a pinene moiety: 40, 41,
53, 54, and 61b. These compounds were also active against
cowpox (SI: 30–406) and mousepox (SI: 39–707) viruses.
1-Adamantanecarboxylic acid amide 40 containing the (−)-10-
amino-2-pinene moiety was the most effective (SI(VV) = 1123,
SI(CPXV) = 406, SI(ECTV) = 707); this compound is promising
for further investigation as a potential antiviral drug for the
treatment of orthopoxvirus infection, including smallpox.
demonstrate higher activity than the acyclic and monocyclic
monoterpene ones. Moreover, bicyclic derivatives show
scientifically lower cytotoxicity leading to an increased SI.
Sulfonamides were found to be more active as well as more
cytotoxic than their structural analogs, derivatives of
camphor. In general, amides containing the 1-adamantanane
fragment were more active than isomeric 2-adamantane
substituted derivatives.
Therefore, initial screening revealed the highest activity
combined with low cytotoxicity in compounds containing
bicyclic monoterpenoid moieties. For further study, we
selected amides containing the (−)-10-amino-2-pinene moiety
(amides 40, 41, 53, 54, and 61b) because this group included
compound 40 that showed the highest SI among the
investigated compounds. These compounds were tested for
activity against cowpox (CPXV, Grishak strain) and mousepox
(ectromelia virus (ECTV), K-1 strain) viruses (Table 2).
Amides 40, 41, 53, 54, and 61b showed high activity
against
the
mousepox
virus.
In
this
case,
1-adamantanecarboxylic acid derivatives (compounds 40 and
41) were more active than 2-adamantanecarboxylic acid
derivatives (compounds 53 and 54) (Table 2). This trend is
observed for all studied orthopoxviruses.
The cytotoxicity of most tested compounds was 2-fold or
lower as compared to the reference drug cidofovir. In this
case, there were compounds whose selectivity index was more
than two orders of magnitude higher than that of the
reference drug, e.g., amide 40 with a SI of 1123, synthesized
based on 1-adamantane carboxylic acid chloride 39 and
(−)-10-amino-2-pinene 23. In our work we didn't investigate
the mechanism of action of the new adamantane derivatives,
since the objective of this study was to demonstrate the anti-
orthopoxvirus activity of our synthesized adamantane–
monoterpene conjugates and to identify the most active ones.
Materials and methods
Chemistry
All chemicals were purchased from commercial vendors and
used without further purification, unless indicated otherwise.
Column chromatography was performed using silica gel (60–
230 μ, Macherey-Nagel) and a solution containing 0% to 5%
ethyl acetate in hexane. Optical rotations [α]TD°C were
measured on a PolAAr 3005 polarimeter, and the optical
rotation values are given in 10−1 deg cm2 g−1. 1H- and 13C-
Table 2 Indicators of cytotoxicity (CC50) and antiviral activity (IC50) of amides 40, 41, 53, 54, and 61b against the cowpox virus (CPXV) and ectromelia
virus (ECTV) in Vero cell culture
CPXV
ECTV
CC50, μM (M
Sm, n = 3)
Compound
IC50, μM (M SD, n = 3)
SI
IC50, μM (M SD, n = 3)
SI
40
41
53
54
1908.6 101.4
750.2 84.6
1225.9 143.9
372.5 97.7
811.5 110.5
4.7 0.2
4.1 0.6
15.0 0.2
12.5 0.1
7.2 0.2
406
183
82
30
113
2.7 0.2
1.6 0.6
12.6 0.2
9.5 0.1
1.7 0.2
707
469
97
39
477
61b
Note: CC50 is the cytotoxic concentration causing 50% cell death in an uninfected monolayer; IC50 is the inhibitory concentration ensuring
50% cell survival in a virus-infected monolayer; as M SD, where M is the mean, and SD is the standard deviation; n = 3 is the number of CC50
and IC50 measurements; SI is the drug selectivity index (CC50/IC50).
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2
NMR spectra were registered on
a
Bruker DRX-500
13-H), 3.78 (1 H, ddm, J = 15.2, J13′,NH = 6.0, 13-H′), 5.32–5.35
(1 H, m, 15-H), 5.45–5.52 (1 H, br m, NH). δC (150 MHz,
CDCl3) 40.58 (s, C-1), 39.26 (t, C-2, C-8, C-9), 28.04 (d, C-3, C-
5, C-7), 34.42 (t, C-4, C-6, C-10), 177.54 (s, C-11), 43.70 (t, C-
13), 144.82 (s, C-14), 118.20 (d, C-15), 31.02 (t, C-16), 40.65 (d,
C-17), 37.82 (s, C-18), 43.90 (d, C-19), 31.39 (t, C-20), 26.00 (q,
spectrometer (500.13 MHz (1H) and 125.76 MHz (13C) in
CDCl3) and on Bruker Avance – III 600 (600.30 MHz (1H) and
150.95 MHz (13C) in CDCl3). Chemical shifts obtained are
given in ppm, relative to residual chloroform (δH 7.24, δC
76.90 ppm), and J values are given in Hz. The compound
structures were determined by analyzing their 1H-NMR
C-21), 21.13 (q, C-22). m/z 313.2396 (M+ C21H31O1N1 , calc.
+
1
1
spectra; H, H double resonance spectra and 2D correlation
313.2400).
1
spectra H–1H (COSY, NOESY); J-modulated 13C-NMR spectra
N-(2-((1R,5S)-6.6-Dimethylbicycloij3.1.1]hept-2-en-2-yl)-
ethyl)adamantane-1-carboxamide 41. Yield 31%. [α]2D4 = −20 (c
1.25 in MeOH). δH (500 MHz, CDCl3) 0.81 (3 H, s, 23-Me),
(JMOD) and 13C,1H-type 2D heteronuclear correlation with
one-bond and long-range spin–spin coupling constants
(COSY and HSQC, J(C,H) = 135 Hz and 145 Hz, respectively,
COLOC and HMBC,2,3 J(C,H) = 10 Hz and 7 Hz, respectively).
Numeration of atoms in the compounds (see the ESI†) is
given for assigning the signals in the NMR spectra and does
not coincide with the nomenclature of the compounds. The
elemental composition of the compounds was determined
from the high-resolution mass spectra (HR-MS) recorded on
a DFS Thermo Scientific spectrometer in full scan mode (0–
500 m/z, 70 eV electron impact ionization, direct sample
injection). The conversion of the reagents, the content of the
compounds in fractions during chromatography and the
purity of the target compounds were determined using gas
chromatography methods: 7820A gas chromatograph (Agilent
Tech., USA), flame-ionization detector, HP-5 capillary column
(0.25 mm × 3 m × 0.25 μm), helium carrier gas (flow rate 2
mL min−1, flow division 99 : 1), temperature range from 120
°C to 280 °C, heating 20 °C min−1. The purity of the target
compounds for biological testing was confirmed to be more
than 95%.
1.08 (1 H, d, J = 8.6, 21-Hanti), 1.24 (3 H, s, 22-Me), 1.62–1.73
2
3
(6 H, m, 4-2H, 6-2H, 10-2H), 1.79 (6 H, d, J = 3.0, 2-2H, 8-2H,
9-2H), 1.97–2.04 (4 H, m, 3-H, 5-H, 7-H, 20-H), 2.05–2.14 (3 H,
m, 14-2H, 18-H), 2.19 (1 H, dm, 2J = 17.7, 17-H), 2.25 (1 H,
dm, 2J = 17.7, 17-H′), 2.36 (1 H, ddd, 2J = 8.6, J21cis,18 = 5.6,
J21cis,20 = 5.6, 21-Hcis), 3.14–3.29 (2 H, m, 13-2H), 5.25–5.28 (1
H, m, 16-H), 5.59 (1 H, br s, NH). δC (125 MHz, CDCl3) 40.41
(s, C-1), 39.12 (t, C-2, C-8, C-9), 28.00 (d, C-3, C-5, C-7), 36.41
(t, C-4, C-6, C-10), 177.57 (s, C-11), 36.36 (t, C-13), 36.30 (t, C-
14), 145.47 (s, C-15), 118.59 (d, C-16), 31.24 (t, C-17), 40.54 (d,
C-18), 37.72 (s, C-19), 45.10 (d, C-20), 31.67 (t, C-21), 26.05 (q,
C-22), 21.12 (q, C-23). m/z 327.2552 (M+ C22H33O1N1 , calc.
+
327.2557).
N-(((S)-4-(Prop-1-en-2-yl)cyclohex-1-en-1-yl)methyl)-
adamantane-1-carboxamide 42. Yield 23%. [α]2D4 = −46 (c 1.60
in MeOH). δH (500 MHz, CDCl3) 1.38–1.48 (1 H, m, 18-Ha),
1.64–1.75 (6 H, m, 4-2H, 6-2H, 10-2H), 1.70 (3 H, s, 22-Me),
1.76–1.82 (1 H, m, 18-He), 1.84 (6 H, d, J = 3.0, 2-2H, 8-2H,
3
9-2H), 1.85–2.14 (8 H, m, 3-H, 5-H, 7-H, 16-2H, 17-H, 19-2H),
3.68–3.79 (2 H, m, 13-2H), 4.66–4.70 (2 H, m, 21-2H), 5.50–
5.54 (1 H, m, 15-H), 5.55–5.62 (1 H, br m, NH).δC (125 MHz,
CDCl3) 40.56 (s, C-1), 39.22 (t, C-2, C-8, C-9), 28.00 (d, C-3, C-
5, C-7), 36.39 (t, C-4, C-6, C-10), 177.68 (s, C-11), 44.54 (t, C-
13), 134.42 (s, C-14), 122.01 (d, C-15), 30.33 (t, C-16), 40.90 (d,
C-17), 27.31 (t, C-18), 26.85 (t, C-19), 149.62 (s, C-20), 108.53
General synthesis procedure
To carboxylic or sulfonic acid chloride (1 eq.) in dry toluene
solution (5 ml) was added triethylamine (1.1 eq.), and the
corresponding amine (1 eq.) in dry toluene (1 ml) was added
to the solution at 0 °C, the resulting mixture was stirred for
12 hours at room temperature. Toluene was evaporated, and
the residue was treated with 10 ml of 5% NaOH (5% KOH in
the case of 58a and 58b) and extracted with EtOAc (3 × 20
ml). The organic layer was washed with 20 ml of saturated
NaCl solution and dried over sodium sulfate and the solvent
was distilled off. The resulting amide was purified by silica
(t, C-21), 20.63 (q, C-22). m/z 313.2402 (M+ C21H31O1N1 , calc.
+
313.2400).
N-((E)-3.7-Dimethylocta-2.6-dien-1-yl)adamantane-1-
carboxamide 44. Yield 29%. δH(600 MHz, CDCl3) 1.58 (3 H, br
s, 21-Me), 1.64 (3 H, br s, 22-Me), 1.66 (3 H, m, 20-Me), 1.64–
3
1.74 (6 H, m, 4-2H, 6-2H, 10-2H), 1.82 (6 H, d, J = 3.0, 2-2H,
8-2H, 9-2H), 1.96–2.03 (5 H, m, 3-H, 5-H, 7-H, 16-2H), 2.03–
2.09 (2 H, m, 17-2H), 3.80 (2 H, br t, J13,14 ≈ 7.0, 13-2H), 5.05
(1 H, tm, J18,17 = 7.0, 18-H), 5.15 (1 H, tm, J14,13 = 7.0, 14-H),
5.43 (1 H, br s, NH). δC(150 MHz, CDCl3) 40.42 (s, C-1), 39.17
(t, C-2, C-8, C-9), 28.03 (d, C-3, C-5, C-7), 36.43 (t, C-4, C-6, C-
10), 177.60 (s, C-11), 37.23 (t, C-13), 120.08 (d, C-14), 139.77
(s, C-15), 39.36 (t, C-16), 26.25 (t, C-17), 123.74 (d, C-18),
131.60 (s, C-19), 25.57 (q, C-20), 17.59 (q, C-21), 16.16 (q, C-
gel
column
chromatography.
The
synthesis
and
characterization of adamantane derivatives 43, 45, 55, 58a,
58b were published previously.36
N-(((1R,5S)-6.6-Dimethylbicycloij3.1.1]hept-2-en-2-yl)-
methyl)adamantane-1-carboxamide 40. Yield 42%. [α]2D5 = −19
(c 0.78 in MeOH). δH (600 MHz, CDCl3) 0.81 (3 H, s, 22-Me),
1.12 (1 H, d, J20anti,20sin = 8.6, 20-Hanti), 1.24 (3 H, s, 21-Me),
3
+
1.64–1.74 (6 H, m, 4-2H, 6-2H, 10-2H), 1.82 (6 H, d, J = 3.0,
22). m/z 315.2554 (M+ C21H33O1N1 , calc. 315.2557).
2-2H, 8-2H, 9-2H), 1.99 (1 H, ddd, J19,17 = J19,20sin = 5.6, J19,15
= 1.5, 19-H), 1.99–2.03 (3 H, m, 3-H, 5-H, 7-H), 2.05–2.09 (1
H, m, 17-H), 2.18 (1 H, dm, J = 17.7, 16-H), 2.25 (1 H, dm, J
N-((S)-3.7-Dimethyloct-6-en-1-yl)adamantane-1-
carboxamide 46. Yield 32%. [α]2D5 = +6 (c 0.87 in MeOH).
δH(600 MHz, CDCl3) 0.87 (3 H, d, J22,15 = 6.6, 22-Me), 1.10–
1.17 (1 H, m, 16-H), 1.22–1.34 (2 H, m, 14-H, 16-H′), 1.38–1.50
(2 H, m, 14-H′, 15-H), 1.57 (3 H, br s, 21-Me), 1.64 (3 H, br s,
2
2
= 17.7, 16-H′), 2.35 (1 H, ddd, J20sin,20anti = 8.6, J20sin,17
=
2
J20sin,19 = 5.6, 20-Hsin), 3.69 (1 H, ddm, J = 15.2, J13,NH = 5.4,
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3
20-Me), 1.63–1.73 (6 H, m, 4-2H, 6-2H, 10-2H), 1.81 (6 H, d, J
= 3.0, 2-2H, 8-2H, 9-2H), 1.87–2.02 (5 H, m, 3-H, 5-H, 7-H, 17-
2H), 3.16–3.27 (2 H, m, 13-2H), 5.04 (1 H, tm, J18,17 = 7.2, 18-
H), 5.51 (1 H, br s, NH). δC(150 MHz, CDCl3) 40.37 (s, C-1),
39.14 (t, C-2, C-8, C-9), 28.00 (d, C-3, C-5, C-7), 36.40 (t, C-4,
C-6, C-10), 177.68 (s, C-11), 37.23 (t, C-13), 36.47 (t, C-14),
30.06 (d, C-15), 36.79 (t, C-16), 25.23 (t, C-17), 124.45 (d, C-
18), 131.20 (s, C-19), 25.57 (q, C-20), 17.54 (q, C-21), 19.30 (q,
N-(((1R,5S)-6.6-Dimethylbicycloij3.1.1]hept-2-en-2-yl)-
methyl)adamantane-2-carboxamide 53. Yield 46%. [α]2D5 = −16
(c 0.62 in MeOH). δH(600 MHz, CDCl3) 0.80 (3 H, s, 22-Me),
1.13 (1 H, d, J20anti,20sin = 8.7, 20-Hanti), 1.24 (3 H, s, 21-Me),
1.57–1.62 (2 H, m, 4-H, 9-H), 1.69–1.77 (4 H, m, 6-2H, 8-H,
10-H), 1.80–1.84 (1 H, m, 5-H or 7-H), 1.84–1.95 (5 H, m, 7-H
or 5-H, 8-H′, 10-H′, 4-H′, 9-H′), 2.04 (1 H, ddd, J19,17 = J19,20sin
= 5.6, J19,15 = 1.4, 19-H), 2.05–2.09 (1 H, m, 17-H), 2.18 (1 H,
+
2
C-22). m/z 317.2717 (M+ C21H35O1N1 , calc. 317.2713).
dm, J = 17.7, 16-H), 2.20–2.28 (3 H, m, 1-H, 3-H, 16-H′), 2.35
N-((1R,2R,4R)-1.7.7-Trimethylbicycloij2.2.1]heptan-2-yl)-
adamantane-1-carboxamide 47. Yield 98%. [α]2D5 = −38 (c 0.94
in MeOH). δH(500 MHz, CDCl3) 0.78 (3 H, s, 20-Me), 0.80 (3
H, s, 21-Me), 0.89 (3 H, s, 22-Me), 1.12 (1 H, ddd, 2J = 12.3,
J16endo,15endo = 9.4, J16endo,15exo = 4.5, 16-Hendo), 1.26 (1 H, ddd,
2J = 12.8, J15endo,16endo = 9.4, J15endo,16exo = 4.1, 15-Hendo), 1.45–
1.56 (2 H, m, 15-Hexo, 18-Hexo), 1.62–1.74 (8 H, m, 4-2H, 6-2H,
(1 H, ddd, J20sin,20anti = 8.7, J20sin,17 = J20sin,19 = 5.6, 20-Hsin),
2
2.42–2.45 (1 H, br s, 2-H), 3.76 (1 H, ddm, J = 15.1, J13,NH
=
2
6.0, 13-H), 3.83 (1 H, ddm, J = 15.1, J13′,NH = 6.0, 13-H′), 5.34–
5.37 (1 H, m, 15-H), 5.45–5.56 (1 H, br m, NH). δC(150 MHz,
CDCl3) 29.81 and 30.00 (2d, C-1, C-3), 49.98 (d, C-2), 33.21
and 33.28 (2t, C-4, C-9), 27.30 and 27.39 (2d, C-5, C-7), 37.26
(t, C-6), 38.25 (t, C-8, C-10), 173.71 (s, C-11), 43.85 (t, C-13),
144.92 (s, C-14), 118.47 (d, C-15), 31.03 (t, C-16), 40.65 (d, C-
17), 37.83 (s, C-18), 43.93 (d, C-19), 31.44 (t, C-20), 25.99 (q,
3
10-2H, 16-Hexo, 17-H), 1.79 (6 H, d, J = 2.7, 2-2H, 8-2H, 9-2H),
2
1.82 (1 H, dd, J = 13.3, J18endo,13endo = 9.1, 18-Hendo), 1.96–2.04
+
(3 H, m, 3-H, 5-H, 7-H), 3.86 (1 H, ddd, J13endo,18endo = 9.1,
C-21), 21.02 (q, C-22). m/z 313.2402 (M+ C21H31O1N1 , calc.
J13,12 = 8.9, J13endo,18exo = 5.0, 13-Hendo), 5.54 (1 H, br d, J12,13
=
313.2400).
8.9, NH). δC(125 MHz, CDCl3) 40.41 (s, C-1), 39.27 (t, C-2, C-8,
C-9), 28.08 (d, C-3, C-5, C-7), 36.44 (t, C-4, C-6, C-10), 176.78
(s, C-11), 55.82 (d, C-13), 48.31 (s, C-14), 35.74 (t, C-15), 26.90
(t, C-16), 44.80 (d, C-17), 39.14 (t, C-18), 46.89 (s, C-19), 11.49
(q, C-20), 20.13, 20.14 (2q, C-21, C-22). m/z 315.2553 (M+
C21H33O1N1+, calc. 315.2553).
N-(2-((1R,5S)-6.6-Dimethylbicycloij3.1.1]hept-2-en-2-yl)-
ethyl)adamantane-2-carboxamide 54. Yield 31%. [α]2D4 = −19
(c 0.93 in MeOH). δH(600 MHz, CDCl3) 0.78 (3 H, s, 23-Me),
1.06 (1 H, d, 2J = 8.7, 21-Hanti), 1.22 (3 H, s, 22-Me), 1.53–
1.58 (2 H, br d, 2J = 12.8, 4-H, 9-H), 1.66–1.73 (4 H, m,
6-2H, 8-H, 10-H), 1.76–1.80 (1 H, m, 5-H or 7-H), 1.81–1.91
(5 H, m, 4-H′, 7-H or 5-H, 8-H′, 9-H′, 10-H′), 2.01 (1H, ddd,
J20,18 = J20,21cis = 5.6, J20,16 = 1.3, 20-H), 2.03–2.07 (1 H, m,
18-H), 2.09–2.24 (6 H, m, 1-H, 3-H, 14-2H, 17-2H), 2.33 (1
H, ddd, 2J = 8.7, J21cis,18 = 5.6, J21cis,20 = 5.6, 21-Hcis), 2.36–
2.39 (1 H, m, 2-H), 3.21–3.33 (2 H, m, 13-2H), 5.23–5.26 (1
H, m, 16-H), 5.64–5.70 (1 H, br m, NH). δC(150 MHz,
CDCl3) 29.75 and 29.80 (2d, C-1, C-3), 49.85 (d, C-2), 33.12
and 33.13 (2t, C-4, C-9), 27.23 and 27.31 (2d, C-5, C-7),
37.19 (t, C-6), 38.19 (t, C-8, C-10), 173.63 (s, C-11), 36.49 (t,
C-13, C-14), 145.38 (s, C-15), 118.35 (d, C-16), 31.17 (t, C-
17), 40.52 (d, C-18), 37.69 (s, C-19), 45.14 (d, C-20), 31.55 (t,
C-21), 26.02 (q, C-22), 20.95 (q, C-23). m/z 327.2560 (M+
N-((1R,2R,4S)-1.3.3-Trimethylbicycloij2.2.1]heptan-2-yl)-
adamantane-1-carboxamide 48. Yield 99%. [α]2D0 = +22 (c 0.56
in MeOH). δHIJ600 MHz, CDCl3) 0.72 (3 H, s, 21-Me), 0.99 (3
H, s, 22-Me), 1.09 (3 H, s, 20-Me), 1.12–1.21 (3 H, m, 17-2H,
19-Hanti), 1.39–1.47 (1 H, m, 16-Hexo), 1.60–1.65 (2 H, m, 16-
Hendo, 19-Hsin), 1.66–1.75 (7 H, m, 4-2H, 6-2H, 10-2H, 15-H),
1.85 (6 H, d, 3J = 2.6, 2-2H, 8-2H, 9-2H), 2.00–2.04 (3 H, m,
3-H, 5-H, 7-H), 3.54 (1 H, dd, J13exo,12 = 9.0, J13exo,17exo = 1.6,
13-Hexo), 5.60 (1 H, br d, J12,13 ≈ 9.0, NH). δC(150 MHz,
CDCl3) 40.85 (s, C-1), 39.48 (t, C-2, C-8, C-9), 28.11 (d, C-3, C-
5, C-7), 36.45 (t, C-4, C-6, C-10), 178.23 (s, C-11), 62.34 (d, C-
13), 38.94 (s, C-14), 48.06 (d, C-15), 25.75 (t, C-16), 27.35 (t, C-
17), 48.21 (s, C-18), 42.42 (t, C-19), 30.60 (q, C-20), 20.87 (q,
C-21), 19.48 (q, C-22). m/z 315.2560 (M+ C21H33O1N1+, calc.
315.2557).
N-(2-((R)-2.2.3-Trimethylcyclopent-3-en-1-yl)ethyl)-
adamantane-1-carboxamide 49. Yield 55%. [α]1D9 = −11 (c 0.4
in MeOH). δH(600 MHz, CDCl3) 0.71 (3 H, s, 21-Me), 0.92
(3 H, s, 20-Me), 1.35–1.43 (1 H, m, 14-H), 1.55 (3 H, m, 22-
Me), 1.55–1.72 (8 H, m, 4-2H, 6-2H, 10-2H, 14-H′, 15-H),
1.80 (6 H, d, 3J = 3.0, 2-2H, 8-2H, 9-2H), 1.80–1.85 (1 H, m,
19-H), 1.97–2.01 (3 H, m, 3-H, 5-H, 7-H), 2.23–2.29 (1 H, m,
19-H′), 3.13–3.20 (1 H, m, 13-H), 3.23–3.30 (1 H, m, 13-H′),
5.16–5.19 (1 H, m, 18-H), 5.62 (1 H, br s, NH). δC(150 MHz,
CDCl3) 40.36 (s, C-1), 39.14 (t, C-2, C-8, C-9), 27.99 (d, C-3,
C-5, C-7), 36.38 (t, C-4, C-6, C-10), 177.62 (s, C-11), 38.68 (t,
C-13), 29.92 (t, C-14), 47.93 (d, C-15), 46.66 (s, C-16), 148.35
(s, C-17), 121.42 (d, C-18), 35.37 (t, C-19), 25.56 (q, C-20),
19.51 (q, C-21), 12.41 (q, C-22). m/z 315.2553 (M+
C21H33O1N1+, calc. 315.2557).
+
C22H33O1N1 , calc. 327.2557).
N-(2-((R)-2.2.3-Trimethylcyclopent-3-en-1-yl)ethyl)-
adamantane-2-carboxamide 56. Yield 33%. [α]1D9 = −11 (c 0.74
in MeOH). δH(600 MHz, CDCl3) 0.74 (3 H, s, 21-Me), 0.95 (3
H, s, 20-Me), 1.39–1.47 (1 H, m, 14-H), 1.58 (3 H, m, all J <
3.0, 22-Me), 1.58–1.67 (3 H, m, 4-H, 9-H, 14-H′), 1.70–1.77 (5
H, m, 6-2H, 8-H, 10-H, 15-H), 1.79–1.95 (7 H, m, 4-H′, 5-H,
7-H, 8-H′, 9-H′, 10-H′, 19-H), 2.22–2.26 (2 H, m, 1-H, 3-H), 2.30
2
(1 H, ddm, J = 15.3, J19′,15 = 7.8, other J ≤ 3.0, 19-H′), 2.41 (1
H, br s, 2-H), 3.20–3.28 (1 H, m, 13-H), 3.32–3.39 (1 H, m, 13-
H′), 5.19–5.22 (1 H, m, 18-H), 5.64 (1 H, br s, 12-H). δC(150
MHz, CDCl3) 29.89 and 29.94 (2d, C-1, C-3), 49.89 (d, C-2),
33.22 (t, C-4, C-9), 27.29 and 27.39 (2d, C-5, C-7), 37.27 (t, C-
6), 38.27 (t, C-8, C-10), 173.90 (s, C-11), 38.73 (t, C-13), 30.11
(t, C-14), 47.90 (d, C-15), 46.72 (s, C-16), 148.44 (s, C-17),
121.46 (d, C-18), 35.36 (t, C-19), 25.64 (q, C-20), 19.59 (q, C-
21), 12.47 (q, C-22). m/z 315.2558 (M+ C21H33O1N1+, calc.
315.2557).
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(1R,5S)-N-(Adamantan-1-yl)-6.6-dimethylbicycloij3.1.1]hept-
2-ene-2-carboxamide 61a. Yield 71%. [α]2D5 = −32 (c 0.37 in
MeOH). δH(600 MHz, CDCl3) 0.79 (3 H, s, 21-Me), 1.11 (1 H,
d, J19anti,19sin = 9.0, 19-Hanti), 1.30 (3 H, s, 20-Me), 1.62–1.70 (6
H, m, 4-2H, 6-2H, 10-2H), 2.00 (6 H, d, 3J = 3.0, 2-2H, 8-2H,
9-2H), 2.03–2.11 (4 H, m, 3-H, 5-H, 7-H, 16-H), 2.30 (1 H, ddd,
1.62 (2 H, br d, 2J = 12.8, 4-H, 9-H), 1.71 (2 H, br s, 6-2H),
2.11 (1 H, dd, J17,16exo = 4.7, J17,18exo = 4.3, 17-H), 2.18–2.24 (1
H, m, 19-Hexo), 2.38 (1 H, ddd, 2J = 18.5, J16exo,17 = 4.7,
2
J16exo,18exo = 3.2, 16-Hexo), 2.96 (1 H, d, J = 15.0, 13-H), 3.35 (1
H, d, 2J = 15.0, 13-H′), 3.63 (1 H, br s, 2-H), 5.65 (1 H, br s,
NH), 1.76–2.05 (13 H, m, signals of unmentioned protons).
δC(150 MHz, CDCl3) 32.84 and 33.52 (2d, C-1, C-3), 58.14 (d,
C-2), 31.16 and 31.24 (2t, C-4, C-9), 26.93 and 26.80 (2d, C-5,
C-7), 37.34 (t, C-6), 37.07 and 37.30 (2t, C-8, C-10), 51.57 (t, C-
13), 59.36 (s, C-14), 216.77 (s, C-15), 42.84 (t, C-16), 42.69 (d,
C-17), 26.91 (t, C-18), 26.76 (t, C-19), 48.57 (s, C-20), 19.82 (q,
2
2J = 19.0, J15,14 = J15,16 = 3.0, 15-H), 2.36 (1 H, ddd, J = 19.0,
J15′,14 = J15′,16 = 3.0, 15-H′), 2.42 (1 H, ddd, J19sin,19anti = 9.0,
J19sin,16 = J19sin,18 = 5.7, 19-Hsin), 2.56 (1 H, ddd, J18,16
=
J18,19sin = 5.7, J18,14 = 1.7, 18-H), 5.35 (1 H, br s, 11-H), 6.20–
6.23 (1 H, m, 14-H). δC(150 MHz, CDCl3) 51.53 (s, C-1), 41.60
(t, C-2, C-8, C-9), 29.37 (d, C-3, C-5, C-7), 36.30 (t, C-4, C-6, C-
10), 166.47 (s, C-12), 144.87 (s, C-13), 127.24 (d, C-14), 31.46
(t, C-15), 40.31 (d, C-16), 37.61 (s, C-17), 41.92 (d, C-18), 31.32
(t, C-19), 25.89 (q, C-20), 20.84 (q, C-21). m/z 299.2242 (M+
C20H29O1N1+, calc. 299.2244).
+
C-21), 19.46 (q, C-22). m/z 365.2015 (M+ C20H31O3N1S1 , calc.
365.2019).
Biology
(1R,5S)-N-(adamantan-2-yl)-6.6-dimethylbicycloij3.1.1]hept-
2-ene-2-carboxamide 61b. Yield 30%. [α]2D7 = −34 (c 0.57 in
MeOH). δH(600 MHz, CDCl3) 0.80 (3 H, s, 21-Me), 1.15 (1 H,
d, J19anti,19sin = 9.0, 19-Hanti), 1.31 (3 H, s, 20-Me), 1.61–1.66 (2
H, m, 4-H, 9-H), 1.70–1.77 (4 H, m, 4-H′, 9-H′, 6-2H), 1.79–
1.86 (6 H, m, 5-H, 7-H, 8-2H, 10-2H), 1.90–1.94 (2 H, m, 1-H,
The vaccinia virus (strain Copenhagen), cowpox virus (strain
Grishak), mousepox virus – ectromelia (strain K-1), obtained
from the State collection of pathogens of viral infections and
rickettsioses of the SRC VB Vector (Koltsovo, Novosibirsk
region, Russia) were used in this work. Viruses were
produced in Vero cell culture in DMEM medium (BioloT,
Russia). The concentration of viruses in the culture fluid was
determined by the method of plaques by titration of the
samples in Vero cell culture, calculated and expressed in
decimal logarithms of plaque-forming units per ml (log10
PFU per ml).43 The concentration of the virus in the samples
used in this work was from 5.6 to 6.1 log10 PFU per ml. The
used series of viruses with the indicated titer was stored at
−70 °C.
2
3-H), 2.09–2.13 (1 H, m, 16-H), 2.33 (1 H, ddd, J = 19.0, J15,14
2
= J15,16 = 3.0, 15-H), 2.39 (1 H, ddd, J = 19.0, J15′,14 = J15′,16
3.0, 15-H′), 2.44 (1 H, ddd, J19sin,19anti = 9.0, J19sin,16 = J19sin,18
=
=
5.7, 19-Hsin), 2.60 (1 H, ddd, J18.16 = J18,19sin = 5.7, J18.14 = 1.7,
18-H), 4.03–4.07 (1 H, m, 2-H), 5.98 (1 H, br d, J11,2 = ≈8.0,
11-H), 6.30–6.33 (1 H, m, 14-H). δC(150 MHz, CDCl3) 31.77
and 31.78 (2d, C-1, C-3), 52.85 (d, C-2), 31.96 and 31.97 (2t, C-
4, C-9), 27.00 and 27.13 (2d, C-5, C-7), 37.42 (t, C-6), 36.98 (t,
C-8, C-10), 166.21 (s, C-12), 144.27 (s, C-13), 127.93 (d, C-14),
31.52 (t, C-15), 40.37 (d, C-16), 37.69 (s, C-17), 42.01 (d, C-18),
31.32 (t, C-19), 25.90 (q, C-20), 20.85 (q, C-21). m/z 299.2242
The
antiviral
activity
of
compounds
against
orthopoxviruses was determined in vitro in Vero cell culture.
The Vero cell monolayer was grown in DMEM (BioloT,
Russia) in the presence of 5% fetal bovine serum (HyClone,
USA) with the addition of gentamicin sulfate (40 units per
ml) and amphotericin B (2.5 units per ml) The same medium
was used as a support for the cultivation of cells with the
virus, but with the addition of 2% serum.
+
(M+ C20H29O1N1 , calc. 299.2244).
N-(Adamantan-1-yl)-1-((1S,4R)-7.7-dimethyl-2-oxobicyclo-
ij2.2.1]heptan-1-yl)methanesulfonamide 63a. Yield 50%. [α]D25
= +26 (c 0.86 in MeOH). δH(600 MHz, CDCl3) 0.88 (3 H, s, 21-
Me), 1.02 (3 H, s, 22-Me), 1.40 (1 H, ddd, 2J = 12.5,
J18endo,19endo = 9.4, J18endo,19exo = 4.0, 18-Hendo), 1.61–1.65 (6 H,
Evaluation of antiviral efficacy of the compounds was
carried out according to an adapted and modified method.25
To assess the effectiveness of each sample 8 three-fold
dilutions were used. The initial concentration of each
compound was 1, 10 or 100 μg ml−1, depending on the
preliminary screening of the antiviral activity and toxicity of
the drug. For evaluating the antiviral activity firstly to the
wells of 96-well plates with a monolayer of cells containing
100 μl of DMEM medium with 2% fetal serum a 50 μl
dilution of the samples was added, and then 50 μl of a 1000
PFU per well virus dose was added. Evaluation of the antiviral
activity was performed after 4 days. After incubation of the
cell monolayers infected with orthopoxvirus and treated with
the tested compounds for 4 days, a vital dye “neutral red”
was added to the culture medium for 1.5 hours. Next, the
monolayer was washed twice with saline solution, the lysis
buffer was added and after 30 min the optical density (OD),
which is an indicator of the number of cells in the monolayer
2
m, 4-2H, 6-2H, 10-2H), 1.84 (1 H, ddd, J = 14.3, J19endo,18endo
=
9.4, J19endo,18exo = 4.8, 19-Hendo), 1.90 (1 H, d, 2J = 18.6, 16-
Hendo), 1.94–2.04 (7 H, m, 2-2H, 8-2H, 9-2H, 18-Hexo), 2.05–
2
2.10 (4 H, m, 3-H, 5-H, 7-H, 17-H), 2.30 (1 H, ddd, J = 14.3,
J19exo,18exo = 11.7, J19exo,18endo = 4.0, 19-Hexo), 2.37 (1 H, ddd, J
2
= 18.6, J16exo,17 = 4.8, J16exo,18exo = 3.2, 16Hexo), 2.98 (1 H, d, J
2
= 15.0, 13-H), 3.46 (1 H, d, 2J = 15.0, 13-H′), 4.99 (1 H, br s,
NH). δC(150 MHz, CDCl3) 55.24 (s, C-1), 43.03 (t, C-2, C-8, C-
9), 29.51 (d, C-3, C-5, C-7), 35.85 (t, C-4, C-6, C-10), 54.31 (t,
C-13), 59.20 (s, C-14), 216.61 (s, C-15), 42.77 (t, C-16), 42.62
(d, C-17), 26.87 (t, C-18), 26.09 (t, C-19), 48.33 (s, C-20), 19.79
+
(q, C-21), 19.59 (q, C-22). m/z 365.2023 (M+ C20H31O3N1S1 ,
calc. 365.2019).
N-(Adamantan-2-yl)-1-((1S,4R)-7.7-dimethyl-2-oxobicyclo-
ij2.2.1]heptan-1-yl)methanesulfonamide 63b. Yield 34%. [α]D25
= +24 (c 2.30 in MeOH). δH(600 MHz, CDCl3) 0.88 (3 H, s, 21-
Me), 1.01 (3 H, s, 22-Me), 1.39–1.45 (1 H, m, 18-Hendo), 1.57–
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that is not destroyed in the presence of the virus, was
measured on an Emax plate reader (Molecular Devices, USA).
The OD values were used to calculate a 50% cytotoxic
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
19 A. Sokolova, O. Yarovaya, M. Semenova, A. Shtro, Y.
Orshanskay,
MedChemComm, 2017, 8, 960.
V.
Zarubaev
and
N.
Salakhutdinov,
The synthesis of the target compounds was carried out under
the state assignment of NIOCH SB RAS. The authors would
like to acknowledge the Multi-Access Chemical Research
Center SB RAS for spectral and analytical measurements. The
study of the antiviral activity of chemical compounds was
carried out under the state assignment of the State Research
Centre of Virology and Biotechnology VECTOR.
20 T. Khomenko, V. Zarubaev, I. Orshanskaya, R. Kadyrova, V.
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